Re: [ccp4bb] problems of co-crystallization of protein-DNA complex

- Protein crystallography

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

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Subject: refinement with REFMAC5
From: Debajyoti Dutta debajyoti_dutta47 {- at -} REDIFFMAIL {- dot -} COM
Date: 2009-08-13

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Subject: Re: problems of co-crystallization of protein-DNA complex
From: Andy Torelli att29 {- at -} CORNELL {- dot -} EDU
Date: 2009-08-13

Subject: Re: problems of co-crystallization of protein-DNA complex
From: Raji Edayathumangalam raji {- at -} BRANDEIS {- dot -} EDU
Date: 2009-08-13

Couple of things, Ru Heng.

1. What buffer conditions is your protein in? Is it similar to the
buffer you describe as using to dissolve your DNA in? In general, you
can even get away with dissolving and annealing the oligos in just
Tris etc.
2. Play with buffer conditions, particularly NaCl concentrations.
3. Tweak the protein and DNA ratios. For nucleosomes, we always got
white precipitate if we did not always titrate the DNA to protein
ratios for every individual prep,

I believe optimization of the above parameters would help with the
white precipitate formation.

Hope that helps.
Raji

-----------
Raji Edayathumangalam
Joint Research Fellow
Brigham and Women's Hospital/
Harvard Medical School
Brandeis University

On Aug 13, 2009, at 12:56 AM, ruheng wrote:

>
> Dear CCP4bbers,
>
> I am now working on a DNA binding protein and the purity of the
> protein is quite good, however the results of DLS showed that the
> protein aggregates terribly in quite a lot of different buffer
> conditions I tried and still no crystals can be obtained. So I am
> going to co-crystallize the protein in complex with DNA. I
> synthesized the oligonucleotides varying different numbers of
> basepairs to determine the optimal length which can bound to my
> protein by EMSA. I dissoved the oligos in the buffer containing
> 50mM Tris-HCl, 100mM NaCl, 10mM MgCl2 and 1mM DTT, pH 7.9 and then
> annealed the DNA into the double stranded form at a final
> concentration of 50uM. When I performed the EMSA experiment, I
> mixed the purified protein with the dsDNA at the molecular ratio
> approximately 1:1, but white precipitate was generated as I mixed
> them.
>
> Does anyone have this kinds of experience when working on DNA
> binding proteins and co-crystallizing the protein-DNA complex? Any
> suggestions from yours will be appreciated.
>
> Thank you all.
>
>
> Ru Heng
>
>
>
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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: refinement with REFMAC5
From: Debajyoti Dutta debajyoti_dutta47 {- at -} REDIFFMAIL {- dot -} COM
Date: 2009-08-13

Next message:
Subject: Re: problems of co-crystallization of protein-DNA complex
From: Andy Torelli att29 {- at -} CORNELL {- dot -} EDU
Date: 2009-08-13